Constitutive androstane receptor (CAR, MB67, NR113) (Baes, M., et al. (1994) Mol. Cell Biol. 14, 1544-1552) is a transcription factor that acts as a xenobiotic sensor, capable of regulating cellular function in response to xenobiotics and endobiotics (di Masi, A., De Marinis, E., Ascenzi, P., and Marino, M. (2009) Mol. Asp. Med. 30, 297-343; Ma, X., Idle, J. R., and Gonzalez, F. J. (2008) Exper. Opin. Drug Metab. Tox. 4, 895-908; and Timsit, Y. E., and Negishi, M. (2007) Steroids 72, 231-246). Activated CAR in tissues heterodimerizes with retinoid X receptor (RXR) and translocates to the nucleus. CAR shares a large portion of its metabolic functions with another member of the NR1 family, pregnane x receptor (PXR, NR112). PXR has structural features in the ligand binding domain (LBD) that allow it to successfully bind a diverse set of chemical motifs. CAR LBD is more compact and yet capable of binding varied structural entities (Wu, B., et al. (2013) Drug Discov. Today 18, 574-581). CAR and PXR bind similar response elements on chromatin and hence regulate an overlapping set of genes (Wei, P., et al. (2002) Pharmacogen. J. 2, 117-126). CAR remains a major player in xenobiotic metabolism by controlling the transactivation of many P450 enzymes and transporters, particularly CYP2B6 and multi-drug resistance 1 (MDR1). CAR is the molecular target of phenobarbital (PB)-induced hepatocellular carcinoma and activation of this receptor is an essential requirement for liver tumor development (Yamamoto, Y., et al. (2004) Cancer Res. 64, 7197-7200; and Huang, W., et al. (2005) Mol Endocrinol. 19, 1646-1653). CAR overexpression in neuroblastoma has been shown to drive doxorubicin resistance by increasing the levels of MDR1 expressed (Takwi, A. A., et al. (2013) Oncogene doi: 10.1038/onc.2013.330). CAR activation in ovarian cancer has been shown to decrease effectiveness of anticancer drugs (Wang, Y., et al. (2014) Biochem. Pharm. 90, 356-366), CAR activation was shown to cause hepatic lipogenesis and insulin insensitivity through upregulation of the thyroid hormone-responsive SPOT14 gene, which might promote fatty liver diseases and insulin resistance (Breuker, C., et al. (2010) Endocrinology 151, 1653-1661). CAR function in various diseases emphasizes the clinical and pharmacological importance of this receptor (e.g., see. Kachaylo, E. M., et al. (2011) Biokhimiia 76, 1087-1097).
There are multiple isoforms of CAR (Auerbach, S. S., et al. (2003) Nucl. Acids Res. 31, 3194-3207). Exogenously expressed hCAR1 spontaneously accumulates in the cell nuclei and tends to be constitutively active in the absence of agonists (Baes, M., et al. (1994) Mol. Cell Biol. 14, 1544-1552). CAR also exhibits high basal but low agonist-induced activation in immortalized cells (Faucette, S. R., et al. (2007) J. Pharm. Exper. Therap. 320, 72-80). The splice variant hCAR3 is inducible by agonists when overexpressed in cells (Auerbach, S. S., et al. (2003) Nucl. Acids Res. 31, 3194-3207; and Chen, T., et al. (2010) J. Pharm. Exper. Therap. 332, 106-115), but the agonist-inducible activity is not substantial without simultaneously overexpressing RXRα (Auerbach, S. S., et al. (2005) Mol. Pharm. 68, 1239-1253). In tissues expressing endogenous hCAR, the receptor is mostly cytoplasmic unless activated by direct binding to ligands such as 6-(4-chlorophenyl)imidazo [2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl) oxime (CITCO) (Maglich, J. M., et al. (2003) J. Biol. Chem. 278, 17277-17283), or binding to indirect ligands such as phenobarbital (Honkakoski, P., et al. (1998) Mol. Cell Biol. 18, 5652-5658). Phenobarbital inhibits the EGF signaling pathway, ultimately resulting in activation of CAR via dephosphorylation at Thr(38) (Mutoh, S., et al. (2013) Sci. Signal 6, ra31). There are few known antagonistic modulators of hCAR, with PK11195 being the most potent of all reported inhibitors (Li, L., et al. (2008) Mol. Pharmacol. 74, 443-453). Clotrimazole, meclizine and androstanol are other moderate inverse agonists of CAR function in in vitro and cell-based transfection assays (Moore, L. B., et al. (2000) J. Biol. Chem. 275, 15122-15127; Huang, W., et al. (2004) Mol. Endocrinol. 18, 2402-2408; Moore, L. B., et al. (2002) Mol. Endocrinol. 16, 977-986). It has been observed that all of these CAR inhibitors are also moderate to potent activators of PXR function (Li, L., et al. (2008) Mol. Pharmacol. 74, 443-453; Moore, L. B., et al. (2002) Mol. Endocrinol. 16, 977-986; Lau, A. J., et al. (2011) J. Pharmacol. Exp. Ther. 336, 816-826; Jones, S. A., et al. (2000) Mol. Endocrinol. 14, 27-39).
In addition to a smaller, unique set of target genes, CAR and PXR co-regulate an overlapping set of metabolizing genes, although the strength of the response at each gene depends on the activation status of both the receptors (Wei, P., et al. (2002) Pharmacogenomics J. 2, 117-126). The CYP2B6 gene, although considered to be a predominantly CAR regulated gene, is also induced equally well by activated PXR (Faucette, S. R., et al. (2006) J. Pharmacol. Exp. Ther. 317, 1200-1209; Faucette, S. R, et al. (2007) J. Pharmacol. Exp. Ther. 320, 72-80). In tissues expressing both PXR and CAR proteins (i.e., the liver or colon) treatment with CAR inhibitors that activate PXR would result in a confused gene expression profile and interpretation of receptor function is confounded by this opposing dual activity of such CAR inhibitors.
Despite several known inhibitors of CAR, the identification of inhibitors that selectively inhibit CAR without activation of PXR has remained elusive. Thus, there remains a need for selective inhibitors of CAR that either simultaneously antagonize PXR, or at least, do not activate PXR. Identification of such selective inhibitors of CAR would be a significant advance for therapeutic treatment of cancer and other diseases. These and other needs are addressed by the present invention.